Vaccination is an effective and economical method of preventing disease, and multivalent, orally administered vaccines will have the widest clinical application. Attenuated Salmonella have been successfully studied in murine models as live vectors for delivery of heterologous antigens to the gastrointestinal immune system. Studies in humans have been limited, and variables important for immunogenicity in humans have not been clearly defined. A Salmonella typhi strain (Ty800) deleted for the phoP/phoQ virulence regulon of Salmonellae is very safe and immunogenic in human volunteers, and this strain has been modified to express clinically relevant heterologous antigens. This proposal describes a program of translational research designed to evaluate Salmonella-based vaccine regimens in humans utilizing bivalent S. typhi vaccine strains. These studies emphasize rational modification of clinically acceptable human vaccine strains and vaccination regimens which may alter immunogenicity of heterologous antigens such as type of antigen, location of antigen within the bacterial cell and mechanism of expression or secretion. Using an established IRB-approved human study protocol, adult volunteers are vaccinated, followed for safety and vaccine shedding, and intensively evaluated for evidence of mucosal, humoral and cellular immune-responses to both S. typhi antigens and the relevant heterologous antigens. A sequential oral vaccination regimen consisting of a S. typhi vaccine strain expressing cytoplasmic Helicobacter pylori urease followed by purified recombinant urease and E. coli heat labile toxin adjuvant will be tested in adult volunteers seronegative for H. pylori. An S. typhi strain expressing a fusion antigen of E. coli hemolysin A linked to urease B which is secreted from the bacterial cell will be studied to determine whether secretion alters immunogenicity in this system. Additional constructs will be generated which express either an E. coli heat labile toxin mutant or an E. coli colonization factor antigen (CFA/I). These new strains will be evaluated in vitro and subsequently tested in volunteers to determine whether these molecules with different structures or subcellular location can engender systemic and/or mucosal immune responses. These studies are designed to provide novel human safety and immunogenicity data which is vital to the clinical development of live bacterial vaccine vectors.